We found that rhabdomyosarcomas show consistently high levels of TGF-beta1, and to a lesser extent TGF-beta3, but lack TGF-beta2 protein by immunohistochemical staining in vivo. We also detected variable levels of TGF-beta protein synthesis in conditioned media of cultured rhabdomyosarcoma (RMS) cells with the mink lung fibroblast (CCL-64) bioassay and the ELISA method. Furthermore, all RMS expressed variable levels of TGF-beta1 mRNA in vitro. These data, in combination with our previous data of TGF-beta-induced inhibition of RMS cell differentiation in vitro, and the known inhibitory effect of TGF-beta in normal myogenesis have suggested to us a role of TGF-beta in human RMS. We have studied levels of TGF-beta1 mRNA expression in RMS cell lines after treatment with 5-azacytodine (AZA), an agent that causes differentiation and inhibition of cell growth in RMS, and found them increased in 2 RMS lines. We then decided to study if there were similar changes with other agents that are known to cause differentiation and growth inhibition, or growth inhibition alone, and selected one RMS cell line (RD) and 3 additional agents, i.e., retinoic acid, TPA, and cytosine arabinoside. The possible role of TGF- beta in the growth and differentiation of this RMS cell line will be pursued in the following ways: (1) Levels of expression and affinity of TGF-beta for its receptors will be studied before and after treatment with the above agents, by binding assays and PAGE of cell lysates incubated with 125I-TGF-beta1. (2) Anti-TGF-beta blocking antibodies will be used along with the agents to determine if their action is linked to levels of TGF- beta1. (3) Nuclear transcription assays will be performed to evaluate if the elevated TGF-beta1 mRNA levels are due to increased transcription rather than decreased turnover. (4) TGF-beta protein levels will be evaluated to determine if they parallel the levels of mRNA.